Protamine neutralization is an essential step for the safe use and inactivation of the unfractionated heparin (UFH) that is widely employed in surgical and non‐surgical procedures involving extracorporeal circulation.
To compare protamine neutralization of different pharmaceutical‐grade UFHs prepared from porcine or bovine intestine (HPI and HBI, respectively). HBI has approximately half the anticoagulant potency of HPI, mostly as consequence of its fraction enriched with N‐sulfated α‐glucosamine disaccharides.
Protamine neutralization of HPI and HBI was evaluated with in vitro, ex vivo and in vivo assays. We also performed in‐depth assessments of the complexation of protamine with these distinct UFHs by using nuclear magnetic resonance and mass spectroscopy.
HPI and HBI interact similarly with protamine on a mass/mass basis; however, HBI requires more protamine than HPI to have its anticoagulant activity fully neutralized, because of its lower potency, which entails the use of higher doses. Nuclear magnetic resonance spectra revealed that HPI precipitates homogeneously with protamine. On the other hand, the low‐sulfated fraction of HBI, enriched with N‐sulfated α‐glucosamine, precipitates at higher concentrations of protamine than the fraction more like HPI, with a preponderance of N,6‐disulfated α‐glucosamine disaccharides. Finally, mass spectroscopy spectra showed that some of the different peptide components of protamine interact preferentially with the heparins, irrespective of their animal origin.
Our results have important medical implications, indicating that protamine neutralization of HBI, determined exclusively by point‐of‐care coagulation assessments, must fail because of its lower‐sulfated fraction with reduced anticoagulant activity that could remain in the circulation after the neutralization procedure.